Illumina NovaSeq 6000 paired end sequencing; ChIP-seq of Helicobacter pylori N6 wild-type and HP1021 deletion mutant under microaerophilic and aerobic conditions (optimal growth and oxidative stress conditions, respectively).
ChIP-seq of Helicobacter pylori N6 wild-type and HP1021 deletion mutant under microaerophilic and aerobic conditions (optimal growth and oxidative stress conditions, respectively).
Bacterial cultures (70 ml BB-FBS) of H. pylori N6 wild-type and ΔHP1021 strains were grown under microaerobic conditions to OD600 of 0.8-1.0 and split into 2 sub-cultures of 35 ml each. The first sub-culture was crosslinked with 1% formaldehyde for 5 min immediately after opening the jar. The second culture was crosslinked after 25-minute incubation under aerobic conditions (air atmosphere, 37 °C, 140 rpm orbital shaking). The crosslinking reactions were stopped by treatment with 125 mM glycine for 10 min at room temperature. The cultures were centrifuged at 4700 × g for 10 min at 4 °C and washed twice with 30 ml of ice-cold 1 × PBS, followed by the same centrifugation step. Samples were resuspended in 1.1 ml IP buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% vol/vol NP-40, 1.0% vol/vol Triton X-100) and sonicated (Ultraschallprozessor UP200s (0.6/50% power, 30 s ON-30 s OFF, ice bucket)) to reach 100-500 bp DNA fragment size. Next, the samples were centrifuged at 12000 × g for 10 min at 4 °C. 100 µl of the supernatant was used for input preparation. 900 µl of the supernatant was incubated with 30 µl of Sepharose Protein A (Rockland, PA50-00-0002) (pre-equilibrated in IP buffer) for 1 h at 4 °C on a rotation wheel. The samples were centrifuged 1000 × g for 2 min at 4 °C; the supernatants were incubated with 40 µg of the HP1021 antibody33 and incubated at 4 °C for 16 h on a rotation wheel. Subsequently, 100 µl of the Sepharose Protein A pre-equilibrated in IP buffer was added to the samples, and the binding reaction was performed for 4 h at 4 °C on a rotation wheel. The samples were centrifuged 1000 × g for 2 min at 4 °C, and the supernatant was discarded. The beads were washed four times with IP-wash buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 % NP-40, 0.1% SDS), twice with TE buffer (10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA), resuspended in 180 µl of TE buffer, and treated with 20 µg/ml RNase A at 37 °C for 30 min. Next, crosslinks were reversed by adding SDS at a final concentration of 0.5% and proteinase K at a final concentration of 20 µg/ml, followed by incubation for 16 h at 37 °C. The beads were removed by centrifugation 1000 × g for 2 min at 4 °C, and the DNA from the supernatants were isolated with ChIP DNA Clean & Concentrator (Zymo Research). Input samples were treated like IP samples, starting from incubation with RNase A. The quality of DNA was validated by electrophoresis in 2% agarose gel, and the concentration was determined with QuantiFluor dsDNA System (Promega). The ChIP-DNA was isolated from three independent H. pylori cultures. The beads were removed by centrifugation 1000 × g for 2 min at 4 °C, and the DNA from the supernatants were isolated with ChIP DNA Clean & Concentrator (Zymo Research). Input samples were treated like IP samples, starting from incubation with RNase A. The quality of DNA was validated by electrophoresis in 2% agarose gel, and the concentration was determined with QuantiFluor dsDNA System (Promega). The ChIP-DNA was isolated from three independent Helicobacter pylori cultures. The DNA library preparation was performed at the Novogene Bioinformatics Technology Co. Ltd. (Cambridge, UK). The DNA fragments were repaired, A-tailed and further ligated with an Illumina adapter. The final DNA library was obtained by size selection and PCR amplification. The library was checked with Qubit and RT-qPCR for quantification and a bioanalyser for size distribution detection.